Abstract
Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a “quick-soak”-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.
Highlights
Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved
All approaches led to crystals that could be phased using the anomalous signal as described in the methods section
CsCl was introduced during all three major steps of sample treatment in crystallography: purification, crystallization, and cryo-protection
Summary
Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. We derived a step-wise procedure encompassing a “quick-soak”-only approach and a combined approach of CsCl supplement during purification and cryo-protection This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. The first and simplest procedure would be a “quick-soak” strategy, using already existing crystals[21] In case this strategy doesn’t yield the means for solving the phase problem, an anomalous scatterer could be introduced at an earlier stage i.e. during protein purification or crystallization. This approach requires the identification of a suitable compound, which is compatible with each of these stages. The structure of this target has so far not been determined by other means
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